Even though the possibility of using cell-free hemoglobin (human and bovine) as a replacement for red blood cells has been considered since the late 19th century, it is only over the last few decades that concentrated efforts have been made in research on blood substitute. The main driving force behind this late effort was the concern regarding the potential transmission of blood borne infectious agents and a worldwide shortage of donor blood (Sellards et al. (1916) J Med Res 34:469; Amberson W R (1934) J Cell Compar Physiol 5:359-382; Amberson W R (1937) Biol Rev 12:48-86; Winslow R M (2002) Curr Opin Hematol 9(2):146-151; Winslow R M (2003) J Intern Med 253:508-517).
In the U.S., the implementation of sensitive screening tests has reduced the risk of infectious disease transmission to 1:63,000 blood transfusions for hepatitis B and 1:493,000 for HIV, with intermediate transmission rates for hepatitis C and human T-cell leukemia virus (Schreiber et al. (1996) N Engl J Med 334 (26):1685-1690). While the question of whether blood can transmit Creutzfeldt-Jacob's disease, or its bovine variant, is yet to be answered, there is still a dramatic improvement in blood safety in the developed world (Hoots et al. (2001) Transfus Med Rev 15(2 Suppl 1):45-59).
On the other hand, the lack of safety of the blood supply in the undeveloped world ought to concern the World Health Organization and the international scientific community on an urgent basis. In developing countries where the infected population is large, an estimated 6 million units of donated blood are not tested for HIV, hepatitis and syphilis (World Health Organization Web Page (2005); Wake et al (1998) Trop Doct 28(1):4-8).
The World Health Organization estimates a worldwide demand for 100 million units of blood per year; every 3.75 seconds a U.S. citizen requires a transfusion. At the same time, the rate of blood donors has fallen. Very often blood banks do not meet the demand because of low donation rates. The U.S. is already importing blood from Europe In the U.S., which annually uses approximately 12 million units, a shortage of 3-4 million units per year has been projected by the year 2030. This projected deficit of donated blood does not take into account the more acute need for blood in natural disasters, terrorist attacks and wars. While the demand for blood is increasing at a rate of 1% per year, U.S. blood donations are decreasing at an annual rate of 1% (Surgenor et al. (1990) N Engl J Med 322(23):1646-1651; Hawkins D. (1999) US News World Rep 126(3):34; National Blood Data Resource Center Web Page (2005); North Central Blood Services Web Page (2005)).
The cost of blood acquisition and testing has dramatically escalated. At present, the cost of collecting, testing and transfusing a unit of blood is about $1,000, and that is without factoring in the costs of lawsuits by those who received screened, but tainted blood (Blumberg et al. (1996) Am J Surg 171:324-330).
Another disadvantage of using red blood cells for transfusion is the fact that they must be kept refrigerated, and even then the packed cells have a shelf life of only 42 days. Also, their transfusion requires blood-typing and cross matching, which cannot be done at the scene of an accident or on a battlefield (Williams et al. (1977) Preservation and Clinical Use of Blood and Blood Components. Hematology. McGraw-Hill Book Company, New York).
2.1. Blood Substitutes
Because of these and other problems in transfusion medicine, it has become necessary to seek a new alternative in blood substitute. An effective blood substitute would eliminate the risk of transfusion-transmitted diseases and change the option available in managing the world's blood supply. A pathogen-free, universally compatible blood substitute without the need for cross matching would open a significant global market for both civilian and military applications. The implications of having a viable oxygen carrying solution are broad, starting with a potentially unlimited blood substitute free of any pathogens. A universal blood substitute could alter emergency treatment procedures for patients in hemorrhagic shock; be used in perioperative hemodilution during elective surgical procedures; prolong the survival time of organs donated for transplantation; improve the blood's oxygen carrying capacity to treat life threatening illnesses such as heart infarcts and strokes; be used in tumor radiosensitization; and in the treatment of anemia and other hematological disorders (Winslow R M. (2002) Curr Opin Hematol 9(2): 146-151).
In less than a decade, blood substitute research has moved from the realm of science fiction to reality. However, the commercial development of a usable blood substitute has been somewhat limited, and not yet successful. Though several different free hemoglobin based blood substitute have been developed, they have been proven unsatisfactory in limited human safety trials because of adverse side effects. The major problem with these products is their vasoconstrictor activity. Other reported problems have been the aggravation of oxidative stress and amplification of systemic inflammatory reactions (Workshop on Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes (1999) NIH, Bethesda Md.; Winslow R M. (2000) Vox Sang 79(1): 1-20).
In fact, the commercial development of HEMASSIST™, Baxter Healthcare, Round Lake, Ill. (U.S. Pat. Nos. 4,598,064 and 4,600,531 to Walder; U.S. Pat. Nos. 4,831,012 and 5,281,579 to Estep) was halted, and the development of HEMOLINK™, a raffinose polymerized human hemoglobin solution, Hemosol, Mississauga, Canada (U.S. Pat. No. 4,857,636 to Hsia) was paused because of the high mortality rate or increase of myocardial infarction in humans. Earlier, the commercial development of OPTRO™, a recombinant Hb, Somatogen, Boulder, Colo. (U.S. Pat. Nos. 5,028,588, 5,563,254 and 5,661,124 to Hoffman et al.; U.S. Pat. No. 5,631,219 to Rosenthal et al.) was canceled because of the serious systemic inflammatory responses observed in the patients tested. HEMOPURE®, bovine glutaraldehyde polymerized hemoglobin solution, Biopure Inc., Cambridge, Mass. (U.S. Pat. Nos. 5,084,558, 5,296,465 and 5,753,616 to Rausch et al.; U.S. Pat. No. 5,895,810 to Light et al.), was put on clinical hold due to the “safety concerns.” In 2002, Northfield Laboratories, Evanston, Ill. (U.S. Pat. Nos. 4,826,811, 5,194,590, 5,464,814, 6,133,425, 6,323,320, and 6,914,127 to Sehgal et al.) failed to get U.S. regulatory approval for its POLYHEME®, a glutaraldehyde polymerized human hemoglobin solution, used in elective surgery patients. Now, POLYHEME® is clinically tested on a compassionate use basis to treat severe hemorrhage of auto accident victims (Workshop on Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes. Sep. 27-29, 1999. NIH, Bethesda, Md.; Simoni J. (2005) In: Artificial Oxygen Carrier. Its Front Line. K. Kobayashi et al. (Eds.). Springer-Verlag, Tokyo 2005 pp. 75-126; Moore E. (2003) J Am Coll Surgeons 196:1-17).
To be effective oxygen carrying plasma expanders, blood substitute must fulfill a number of requirements. In addition to being pathogen-free, non-toxic, non-immunogenic, and non-pyrogenic and having an extended shelf-life, these products should have a satisfactory oxygen carrying capacity close to that of whole blood, sufficient to permit effective tissue oxygenation and the circulatory retention time of at least 24 hours. The colloid osmotic pressure and viscosity of the blood substitute product should not exceed those of plasma (Workshop on Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes (1999) NIH, Bethesda, Md.; Guidance for Industry. Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes (2004) U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologies Evaluation and Research, Rockville, Md.).
The effective blood substitute besides being able to immediately maximize blood flow (vasodilation) and tissue/organ perfusion (oxygenation), these products should also stimulate erythropoiesis. Since the circulatory retention time of blood substitute is short (half-life of less than 24 hours) and the heme autoxidation rate is high (more than 30% per day), the erythropoietic activity of these products is an essential component in blood loss anemia treatment. The oxidized heme looses its ability to transport oxygen, therefore the stimulation of erythropoiesis becomes an extremely important element of treatment with blood substitute. A speedy replacement of blood loss with the endogenous red blood cells seems to be the most attractive future of blood substitute. In another words, in a treatment of acute anemia the blood substitute should work as a temporary “oxygen” bridge until the body will be able to produce enough red blood cells to maintain proper tissue oxygenation. (Workshop on Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes (1999) NIH, Bethesda, Md.).
Erythropoiesis, an integral part of hemopoiesis, is the development of red blood cells from pluripotent stem cells through several stages of cell division and differentiation. The pluripotent stem cell gives rise to myeloid stem cells (CFU-GEMM) that turn into the burst-forming units erythroid cells (BFU-E), then into the colony forming unit erythroid cells (CFU-E) and pro-erythroblasts. A pro-erythroblast turns into basophilic normoblast and polychromatophilic erythroblast as the cell begins to produce hemoglobin. Then into the ortochromatic erythroblast when the cytoplasm becomes more eosinophilic. After extruding its nucleus the cells enter circulation as reticulocytes and within a few days become mature red blood cells after loosing their polyribosomes. In normal conditions, the entire erythropoietic process should take no more than 5 days. The life span of mature red blood cells is approximately 90-120 days and requires their continuous replacement (Hillman R S, Finch C A: Red Cell Manual. 7th ed., Philadelphia: F. A. Davis, c1996, viii, 190 pp.).
The regulation of erythropoiesis is a complex process controlled by a highly sensitive feedback system based on oxygen tension (concentration) and the cellular redox-state that involves oxygen and redox regulated transcription factors and many growth factors (erythropoietin-EPO, IL-3, IL-9, SCF, GM-CSF) and minerals, particularly iron (Adamson J W (1991) Biotechnology 19:351-361; Sasaki R (2003) Int Med 42(2): 142-149; Lacombe C et al. (1998) Haematologica 83(8):724-732).
While EPO is not the sole growth factor responsible for erythropoiesis, it is the most important regulator of the proliferation of committed progenitors and an anti-apoptotic protector. A principal function of EPO through the EPO receptor (EpoR) is to rescue the committed erythroid progenitors from apoptosis. EPO-dependent upregulation of the antiapoptotic protein Bcl-X(L) allows “default” terminal differentiation of apoptosis-protected, committed erythroblasts, independent of any exogenous signals (Socolovsky et al. (1999) Cell 98(2):181-91; Dolznig et al. (2002) Curr Biol 12(13):1076-1085).
A schematic representation of erythropoietic events and factors involved: CFU-GEMM→(EPO, SCF, IL-9)→BFU-E→(EPO, SCF, GM-CST, IL-3)→CFU-E→(EPO, GM-CSF)→ERYTHROBLAST (EPO)→RETICULOCYTE→RBC
Anemia is defined as a pathologic deficiency of oxygen-carrying capacity of blood, resulting in hypoxia. The main causes of anemia are acute blood loss, chronic illnesses secondary to refractory anemia, cancer, intravascular hemolysis, and increase in red blood cell sequestration or decrease of its production. A natural response to hypoxia is an increase in the erythropoietic response (Campbell K. (2004) Nurs Times 100(47):40-43).
Hypoxia simulates the peritubular interstitial cells of the kidney (cortex) to produce EPO. EPO is also synthesized in the liver, and astrocytes in the brain where it protects against neuronal apoptosis and damage during hypoxia. Oxygen-regulated transcription factors; hypoxia inducible factor-1 alpha (HIF-1 alpha) and -1 beta (HIF-1 beta) mediate this process which goes under the control of a single gene on human chromosome 7. HIF-1 that binds specifically to the 3′ enhancer of the gene encoding EPO is also a promoter in other genes important in adaptation to hypoxia (Semenza et al. (1992) Mol Cell Biol 12; 5447-5454; Brines et al. (2005) Nat Rev Neurosci 6(6):484-494).
HIF-1, identified by Semenza & Wang, is a heterodimer composed of two basic helix loop-helix/PAS proteins (HIF-1 alpha) and the acryl hydrocarbon nuclear translocator HIF-1 beta. HIF-1 beta is not affected much by oxygen, whereas HIF-1 alpha is present only in the hypoxic condition. In normoxia, the degradation of HIF-1 alpha depends on oxygen mediated hydroxylation of its proline residues by pyrol-4-hydroxylase. Hydroxylation of HIF-1 alpha initiates its rapid degradation by the von Hippel-Lindau tumor suppressor protein that binds to the hydroxylated but not to the non-hydroxylated domain. The von Hippel-Lindau tumor suppressor protein is a part of an ubiquitin ligase linking HIF-1 alpha to the ubiquitination machinery (Wang et al. (1995) J Biol Chem 270; 1230-1237; Wang et al. (1993) J Biol Chem 268; 21513-21518; Kallio et al. (1999) J Biol Chem 274(10):6519-6525).
In the hypoxic condition, however, a lack of oxygen suppresses the degradation of HIF-1 alpha, which rapidly translocates from the cytoplasm to the nucleus and acts as a master regulator of several dozens of oxygen-regulated target genes involved in:
1) oxygen transport: erythropoiesis (EPO); iron transport (transferrin); iron uptake (transferrin receptor),
2) vascular regulation: angiogenesis (VEGF, EG-VEGF, PAI-1); control of vascular tone (iNOS, alpha 1 B-adrenergic receptor, ET-1); vascular remodeling (HO-1),
3) anaerobic energy: glucose uptake (glucose transporter 1); glycolysis regulation (PFKFB3); glycolysis (phosphofructokinase 1, aldolase, GAPDH, phosphoglycerate kinase 1, enolase 1, lactate dehydrogenase A (Wenger R H (2002) FASEB J 16; 1151-1162; Gleadle et al. (1997) Blood 89(2):503-509; Gleadle et al. (1998) Mol Med Today 4(3):122-129).
HIF-1 alpha can also be stabilized in normoxia. For instance, in oxidative stress the reactive oxygen species (ROS) by changing the cellular redox equilibrium that activates NF-kappa B and induces inflammatory genes (i.e., TNF-alpha, IL-1 beta, IL-6), may stabilize HIF-1 alpha. These inflammatory cytokines, however, can also inhibit HIF-1 alpha binding to the EPO gene while promoting VEGF gene induction, thus suppressing erythropoiesis and accelerating angiogenesis. In cancer patients this phenomena may result in severe anemia and excessive tumor growth, due to effective angiogenesis. Similarly, an other inflammatory mediator TGF-beta, which is also known to stabilize HIF-1 alpha under normoxic conditions, is capable of blocking the differentiation of erythroid progenitor cells while decreasing EPO's erythropoietic activity (Hellwing-Burgel et al. (1999) Am Soc Hematol 94:1561-1567; Linch D C (1989) Schweiz Med Wochenschr 119(39): 1327-1328).
Inflammation is also implicated in the pathogenesis of EPO resistance in patients with end-stage renal disease. TNF-alpha, IL-1 beta and IL-6 are suggested to suppress erythropoiesis in uremia. In animal models and in humans, administration of IL-6 causes a hypoproliferative anemia by direct inhibition upon erythroid progenitor cells (Trey et al. (1995) Crit Rev Oncol Hematol 21:1-8; Yuen et al. (2005) ASAIO J 51(3):236-241).
Other factors known to stabilize HIF-1 alpha under normoxic conditions include NO, PDGF, and oxLDL. The molecular pathways that govern HIF-1 alpha normoxic regulation is mediated by ROS, PI3K, TOR and MAP kinases, particularly ERK 1/2 (Haddad et al. (2000) J Biol Chem 275(28):21130-21139; Haddad et al. (2001) FEBS Lett 505(2):269-274; Lando et al. (2000) J Biol Chem 275(7):4618-4627; Richard et al. (1999) J Biol Chem 274(46):32631-32637).
The first observation about the possible involvement of free hemoglobin in erythropoietic responses came in 1949 from Amberson, who observed an increase in erythropoiesis indicators (reticulocyte count and hematocrit) in a human after administration of crude hemoglobin solution. This experiment ended tragically with patient death due to the renal failure (Amberson et al. (1949) J Appl Physiol 1:469-489). A clinical trial conducted 30 years later by Savitsky who infused stroma-free hemoglobin solution into normal human volunteers had a similar tragic consequence. All subjects treated with this hemoglobin showed systemic hypertension and renal failure, while one person died (Savitsky et al. (1978) Clin Pharmacol Ther 23:73-80).
These early clinical experiments proved that uncross-linked hemoglobin is deadly and not suitable for transfusion. At that time the authors have been unable to explain the mechanism of these pathological events. By applying current knowledge, it is reasonable to suggest that the pathological responses seen in Amberson's and Savitsky's clinical trials, particularly, rapid rise in blood pressure, was a result of intrinsic toxicity of hemoglobin. Now, it is obvious that hemoglobin-based blood substitute by scavenging nitric oxide and affecting other vascular tone controlling-mechanisms can produce severe rise in blood pressure which is associated with decreased cardiac output and increased total vascular peripheral resistance (Simoni J. (2005) In: Artificial Oxygen Carrier. Its Front Line. K. Kobayashi et al. (Eds.). Springer-Verlag, Tokyo, 2005 pp. 75-126).
Hemoglobin is a pressor agent and the presently used chemical or recombinant modification techniques did not correct this problem. All tested blood substitute products, including HEMASSIST™, OPTRO™-rHb1.1, POLYHEME®, HEMOPURE® and HEMOLINK™ caused vascular constriction—a side effect that has been the main nemesis of blood substitute developers. The observed increase in blood pressure after injection of these blood substitute is caused by an increase in peripheral vascular resistance resulting from vasoconstriction (Winslow R M (1994) Transf Clin Biol 1(1); 9-14; Hess et al. (1994) Artif Cells Blood Substit Immobil Biotechnol 22(3):361-372; Kasper et al. (1996) Cardiovasc Anesth 83(5):921-927; Kasper et al. (1998) Anesth Anal 87(2):284-291; Winslow R M (2003) J Intern Med 253:508-517). In was also reported that some of the products have a tendency to shut down capillary flow, which may decrease the tissue/organ perfusion rate and produce hypoxia (Cheung et al. (2001) Anesth Anal 93(4):832-838).
Since the hypoxic environment stabilizes HIF-1 alpha, it is theoretically possible that blood substitute that promote vasoconstriction and produce hypoxia might induce HIF-1 alpha regulated genes. This mechanism, however, is in contradiction to the main role for blood substitute, which is delivery of a sufficient amount of oxygen to the tissues gasping for air. The proper delivery of oxygen to ischemic organs is a principal requirement in the regulatory approval of hemoglobin solutions as blood substitute. Therefore such an “erythropoietic effect” should be considered pathological. To be considered non-toxic and efficacious, blood substitute products should maximize blood flow and tissue perfusion and therefore, oxygenation (Workshop on Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes (1999) NIH, Bethesda, Md.; Guidance for Industry. Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes (2004) U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologies Evaluation and Research, Rockville, Md.).
It is also theoretically possible, that such blood substitute, when used in larger doses, can trigger inflammatory reactions which might inhibit their initial, hypoxic-driven erythropoietic response.
In the 1990's, Simoni et al. discovered that hemoglobin is a potent inducer of the redox regulated transcription factor NF-kappa B that is involved in the regulation of genes involved in inflammation. He found that the activation of the endothelial NF-kappa B might be dependent on hemoglobin's pro-oxidant potential and the extent of hemoglobin-mediated cellular oxidative stress that shifts GSH/GSSG into an oxidative equilibrium. In this study, the glutaraldehyde polymerized bovine hemoglobin appeared to be a more potent inducer of NF-kappa B than unmodified hemoglobin. Simoni et al. linked this effect with the fact that glutaraldehyde polymerized hemoglobin produced the highest endothelial lipid peroxidation and the largest depletion of intracellular GSH. Based on these studies, Simoni et al. suggested that the activation of NF-kappa B could be considered as a “bridge” between hemoglobin-induced oxidative stress and hemoglobin-mediated inflammatory responses. Besides, his discovery established the foundation for seeing hemoglobin as a signaling molecule (Simoni et al. (1997) Artif Cells Blood Substit Immobil Biotechnol 25(1-2): 193-210; Simoni et al. (1997) Artif Cells Blood Substit Immobil Biotechnol 25(1-2):211-225; Simoni et al. (1998) ASAIO J 44(5):M356-367; Simoni et al. (1994) Artif Cells Blood Substit Immobil Biotechnol 22(3):525-534; Simoni et al. (2000) ASAIO J 46(6):679-692; Simoni et al. (1994) Artif Cells Blood Substit Immobil Biotechnol 22(3):777-787; Pahl H L (1999) Oncogene 18:6853-6866; Gilmore T D (2005)).
Subsequent research by Simoni, established evidence that hemoglobin solutions which trigger NF-kappa B may suppress HIF-1 alpha regulated genes, particularly EPO (Simoni et al (2003) Artificial Blood 11(1):69; Simoni et al. (2003) ASAIO J 49(2):181; Simoni J. (2005) In: Artificial Oxygen Carrier. Its Front Line. K. Kobayashi et al. (Eds.). Springer-Verlag, Tokyo, 2005 pp. 75-126).
It was also reported that high activity of the NF-kappa B pathway in early erythroid progenitors is involved in the suppression of erythroid-specific genes (Liu et al. (2003) J Biol Chem 278(21):19534-19540).
Inflammation is generally accepted to contribute to the pathogenesis of EPO resistance, particularly in anemia and cancer (Yuen et al. (2005) ASAIO J 51 (3):236-241; Hellwig-Burgel et al. (1999) 94(5):1561-1567).
Therefore, it is theoretically possible that blood substitute, which change the cellular redox state, might trigger NF-kappa B regulated genes (i.e., cytokines) and stabilize HIF-1 alpha even in the normoxic environment. However, in such a condition, effective binding of HIF-1 alpha to the EPO gene is inhibited by the inflammatory cytokines. Since hemoglobin is linked to the production of inflammatory cytokines, and the inflammatory cytokines are potent anti-erythropoietic agents, it is reasonable to suggest that the blood substitute with high pro-inflammatory potential could inhibit the erythropoietic responses.
In fact, it was reported that some of the currently tested blood substitute mediate not only vasoconstrictive events, but they are also capable to induce inflammatory reactions. Those responses were more evident during a dose escalation study (late phases of clinical trials) and have been observed with Baxter's HEMASSIST™, Biopure's HEMOPURE®, Somatogen's rHb1.1-OPTRO™ and Northfield's POLYHEME®. The vasoconstriction and ischemic/inflammatory responses were cited as the main reason for redirecting, halting, or discontinuing the clinical development of these blood substitute (Simoni J. (2005) In: Artificial Oxygen Carrier. Its Front Line. K. Kobayashi et al. (Eds.). Springer-Verlag, Tokyo, 2005 pp. 75-126).
Another scientific rationale against the possible erythropoietic activity of the currently tested blood substitute is the fact that hemoglobin has a natural pro-apoptotic potential. This observation is very important since the principal function of EPO as a pro-erythropoietic agent is to protect committed erythroblasts from apoptosis, thus allowing erythropoiesis to happen (Socolovsky et al. (1999) Cell 98(2): 181-91; Dolznig et al. (2002) Curre Biol 12(13): 1076-1085).
It was reported that unmodified hemoglobin has pro-apoptotic potential toward human endothelial cells and that caspase-8 and -9 controls this effect, which can be accelerated by depletion of intracellular GSH (Meguro et al. (2001) J Neurochem 77(4): 1128-1135; Simoni et al. (2002) ASAIO J 48(2): 193). A diaspirin modified hemoglobin (HEMASSIS™) and its glutaraldehyde-polymerized version also induces morphological changes, G2/M arrest, and DNA fragmentation, indicative of apoptotic cell death (Goldman et al. (1998) Am J Physiol 275(3 Pt2):H1046-53); D'Agnillo et al. (2001) Blood 98(12):3315-3323). Oxyglobin, a veterinary version of HEMOPURE® (Biopure), used in ex vivo heart perfusion model, was found to produce apoptotic endothelial cells death (TUNEL assay) that was associated with a significant increase in coronary artery resistance (Mohara et al. (2005) ASAIO J 51(3):288-295).
It became evident that any agent with pro-apoptotic potential that has direct contact with the bone marrow cells has anti-erythropoietic activity. Such an agent will compete with the anti-apoptotic effects of EPO, making erythropoiesis impossible. In fact, hemoglobin based blood substitute are partially cleared up by the bone marrow cells, therefore, they are in a direct contact with erythroblasts (Shum et al. (1996) Artif Cells Blood Substit Immobil Biotechnol 24(6):655-683).
Realizing that hemoglobin has pro-apoptotic potential and can have direct contact with the bone marrow, it is reasonable to suggest that any blood substitute product with pro-apoptotic activity when given in a relatively high concentration will inhibit erythropoiesis.
In scientific and patent literature, there is limited information about the erythropoietic potency of hemoglobin-based blood substitute. In 1997, Rosenthal et al. (U.S. Pat. No. 5,631,219) claimed the method for stimulation of hemopoiesis in a mammal with the recombinant hemoglobin (rHb1.1) through enhancing growth or differentiation of progenitor stem cells including erythroid progenitor cells. U.S. Pat. Nos. 5,028,588, 5,563,254 and 5,661,124 to Hoffman et al. protects the recombinant hemoglobin rHb1.1 (trade name OPTRO™).
In U.S. Pat. No. 5,631,219, rHb1.1 in a dose of either 0.5 or 1.0 mg/kg body weight, given intravenously three times per week to mice, resulted in a increased BFU-E that are early precursors of red blood cells in the bone marrow. In U.S. Pat. No. 5,631,219, Rosenthal et al. reported an increase in hematocrit following the treatment of normal mice (BDF-1) with rHb1.1. To evaluate whether rHb1.1 acted at a level other than the committed erythroid precursor, Rosenthal evaluated the influence of rHb1.1 on very early, uncommitted progenitor cells, the colony forming unit-spleen (CFU-S).
According to U.S. Pat. No. 5,631,219, Rosenthal found that rHb1.1 in a concentration of 0.5 mg/kg body weight increases the number of CFU-S. In U.S. Pat. No. 5,631,219, the lower dose level of 0.5 mg of hemoglobin/kg body weight appears to work better than the higher doses of rHb1.1 (5 and 10 mg/kg), suggesting a maximum effect at unexpectedly low doses.
Without providing any theoretical explanation, Rosenthal et al. concluded that rHb1.1 at low doses (0.5 mg/kg body weight) works either directly on progenitor cells or indirectly to enhance hematopoiesis and acts as an erythropoietic factor.
The concentration of rHb1.1 used by Rosenthal et al. (0.5-10 mg/kg body weight) was clinically irrelevant in respect to oxygen transport, therefore the treatment of acute blood loss. To be therapeutically effective, hemoglobin-based blood substitute should be transfused in grams, but not milligrams. Therefore, the U.S. Pat. No. 5,631,219 could not apply to the treatment of acute blood loss anemia.
Furthermore, the patented claims in U.S. Pat. No. 5,631,219 are in disagreement with the examples provided. Rosenthal has claimed the therapeutically effective level of hemoglobin to be between 0.001 and 10,000 mg/kg body weight. These claims are not supported by Rosenthal's examples that showed that rHb1.1 in a concentration of only 0.5 mg/kg body weight had hemopoietic effect. Perhaps, Rosenthal was influenced by our published paper in which chemically modified bovine hemoglobin solution in a dose of approximately 1.75 g (1,750 mg)/kg body weight showed an effective erythropoietic response in man. U.S. Pat. No. 5,631,219 relies on this paper (Feola et al. (1992) Surg Gynecol Obstet 174(5):379-386).
In 1997, Moqattash et al. compared the ability of infused rHb1.1 and EPO to rescue the hematopoietic activity from the suppressive effects of AZT in normal and AIDS mice. The result showed that higher concentrations of rHb1.1 used (10-15 mg/kg body weight) did not result in a more significant increase in most blood indices. Moreover, the combination treatment, 5-mg rHb1.1/kg body weights plus 2 U EPO/mouse/day, was showed to work better than 5-mg/kg-body weight of rHb1.1 alone (Moqattash et al. (1997) Acta Haematol 98(2):76-82).
Two years later, Lutton et al. successfully challenged Rosenthal's work. By analyzing the hematopoietic effect of clinically relevant doses of cross-linked and non-cross-linked hemoglobin in rabbits, he concluded that both hemoglobin solutions at high concentrations did not produce a significant variation in the generation of BFU-E and CFU-S, thus, they do not represent any hemopoietic activity (Lutton et al. (1999) Pharmacology 58:319-324).
The recombinant (i.e., rHb1.1), cross-linked tetrameric (i.e., HEMASSIST™), and polymerized (i.e., HEMOPURE®, POLYHEME®) hemoglobins have been extensively tested in various preclinical and clinical studies. All tested hemoglobin solutions showed to be toxic (Workshop on Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics as Red Cell Substitutes (1999) NIH, Bethesda, Md.).
The human clinical trial with rHb1.1 in which 48 healthy male volunteers were randomly assigned to receive 15-320 mg/kg body weight of 5% rHb1.1 was associated with serious side effects, such as gastrointestinal upset, fever, chills, headache, and backache (Viele et al. (1997) Anesthesiology 86(4):848-858). In another clinical study with the patients undergoing surgery and receiving 67-365 mg/kg body weight of rHb1.1, no serious adverse events occurred. However, patients suffered from hypertension, inflammatory symptoms and elevated pancreatic enzymes. In these clinical trials, the erythropoietic effects of rHb1.1 were also not reported (Hayes et al. (2001 Cardiothorac Vase Anesth 15(5):593-602).
Highly unsatisfactory clinical experience with rHb1.1 had ended the commercial development of this recombinant blood substitute product. In the late 90's, Somatogen/Baxter focused on a novel second-generation recombinant product (rHb2.0; U.S. Pat. No. 6,022,849 to Olson et al.) to replace the clinically unsuccessful rHb1.1. The new product was designed to have a lower rate of reaction with nitric oxide. However, after 2 years of pre-clinical testing, the commercial development of rHb2.0 was also discontinued.
Clinical experience with rHb1.1 can help understand why in U.S. Pat. No. 5,631,219 the lower dose level (0.5 mg of hemoglobin/kg body weight) appeared to work better than the higher doses (5 and 10 mg/kg). Perhaps, strong pro-inflammatory and pro-apoptotic potential of the higher doses of rHb.1.1 suppresses the induction of the EPO gene, making erythropoiesis impossible. Therefore, it is reasonable to suggest that rHb1.1 in higher concentrations than a few mg/kg body weight would produce the inhibition of erythropoiesis, while promoting the production of pro-inflammatory phagocytes as a part of the hemopoietic-inflammatory event.
Other hemoglobin based blood substitute products were also unsuccessful in late phases of clinical trials.
Phase III studies with HEMASSIS™ ended tragically. The patients treated with HEMASSIST™ had significantly higher mortality rates than those of the control group. In June 1998, upon recommendation of the FDA, the development program of HEMASSIST™ was suspended due to safety concerns. In HEMASSIST™ clinical trials, the erythropoietic or hemopoietic effect was not reported (Sloan et al. (1999) JAMA 282:1857-1864).
The clinical development of HEMOPURE® (U.S. Pat. Nos. 5,084,558, 5,296,465 and 5,753,616 to Rausch et al.; U.S. Pat. No. 5,895,810 to Light et al.) was put on clinical hold due to “safety concerns.” The strong vasoconstrictive, pro-oxidant, pro-inflammatory and pro-apoptotic potential of this product could inevitably limit its practicability as a blood substitute (Kasper et al. (1996) Cardiovasc Anesth 83(5):921-927; Kasper et al. (1998) Anesth Anal 87(2):284-291).
The erythropoietic effect of HEMOPURE® alone was never substantiated (Gawryl M S (2003) Artif Blood 11(1):46). However, HEMOPURE® (plus EPO) was used experimentally in the treatment of severe anemia after gastrointestinal hemorrhage in a Jehovah's Witness. A 50-yr-old man with initial hemoglobin of 3.5 g/dL was injected with HEMOPURE® (7 units) and with a high-dose of recombinant EPO (500 U/kg/day). Hemoglobin levels were initially maintained and then slowly increased to a maximum of 7.6 g/dL on day 24 of rEPO therapy. This case demonstrates that HEMOPURE® (with a half life less than 24 hours) can serve as initial therapy while awaiting the maximal effect of recombinant EPO on bone marrow red blood cell production. This clinical study showed that HEMOPURE® alone does not have erythropoietic potential (Gannon et al. (2002) Crit Care Med 30(8):1893-1895).
In 2002, POLYHEME® (U.S. Pat. Nos. 4,826,811, 5,194,590, 5,464,814, 6,133,425, 6,323,320 and 6,914,127 to Sehgal et al.) failed to receive U.S. regulatory approval for use in elective surgery patients. Now, POLYHEME® is clinically tested on a compassionate use basis to treat severe hemorrhage of auto accident victims.
In the past POLYHEME® was used to treat a critically anemic woman who suffered from persistent colonic bleeding and hemoglobin of 2.9 g/dL. In this clinical study, POLYHEME® was used together with high dose of recombinant EPO, which was needed to stimulate erythropoietic responses. Therefore, it is highly probable that POLYHEME®, similarly to HEMOPURE®, does not alone have any erythropoietic activity (Allison et al. (2004) Southern Med J 97(12): 1257-1258).
The clinical development of HEMOLINK™ (U.S. Pat. No. 4,857,636 to Hsia) was halted because of increased myocardial infarction (inflammation-based) rates in humans. HEMOLINK™ was shown to be less stable in respect to autoxidation, oxidative modification, and the integrity of the heme group compared to native hemoglobin. HEMOLINK™ that represents high vasoconstrictive, pro-oxidative and pro-inflammatory properties was never characterized as a product that stimulates erythropoiesis alone (Alayash Al (2004) Nature 3:152-159; Riess J G (2001) Chem Rev 101(9):2797-2919).
In the past, similarly to other blood substitute products (HEMOPURE®, POLYHEME®), HEMOLINK™ was tested in compassionate treatment of a 53-yr-old female Jehovah's Witness with severe anemia and hemoglobin of 3.2 g/dL. Also in this trial, HEMOLINK™ was administrated along with a high dose of recombinant EPO and ferrous sulfate. After 14 days, the patient's hemoglobin level increased to only 6.5 g/dL with a hematocrit of 23%. This trial provided more evidence that toxic hemoglobin based blood substitute could not alone stimulate erythropoietic events (Lanzinger et al. (2005) Can J Anaesth 52(4):369-373).
The basic research on erythropoietic activity of hemoglobin is also very limited. Recently, it was reported that hemoglobin under hypoxic conditions increased the expression of HIF-1 alpha. Using a bovine aortic endothelial cell model and the Western Blot method for the detection of HIF-1 alpha it was suggested that the higher expression of HIF-1 alpha is connected with the loss of ferrous- and accumulation of ferric-Hb (oxidation of heme), in both unmodified hemoglobin solutions. In this study, the authors used the diaspirin cross-linked hemoglobin, similar to that of HEMASSIST™ (Yeh et al. (2004) Antioxid Redox Signal 6:944-953).
This experiment besides providing more molecular details for an earlier suggestion that prolonged exposure of endothelial cells to ferric-(oxidized) but not ferrous-(oxygenated) hemoglobin renders these cells remarkably resistant to the secondary oxidant challenge via increased production of HO-1 and ferritin, also suggest that the phenomenon is now known to be HIF-1 alpha regulated (Balla et al. (1995) Am J Physiol 268(2 Pt 1):L321-327).
Because efficacious hemoglobin-based oxygen carriers must be able to counteract the hypoxic conditions associated with blood loss anemia, the above findings may only apply to those products that aggravate hypoxia, thus inducing HIF-1 alpha. In fact, the blood substitute products under current clinical development possessed well-documented vasoconstrictive potential and high autoxidation rate. In Yeh's work, however, no connection between hemoglobin and erythropoiesis has been made.
An evident lack of erythropoietic activity of blood substitute under current development can be summarized by the statement of Dr. Harvey G. Klein from the Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institute of Health, Bethesda, Md. In his 2005 review paper entitled: “Blood substitutes: how close to a solution?” he stated that: “ . . . hemoglobin-derived red cell substitutes from human, bovine and recombinant sources in phase III trials all have a half-life measured in hours and are unlikely to replace transfusions or drugs that stimulate erythropoiesis for chronic anemia, but they may play role: (1) as a bridge to transfusion when no compatible blood is immediately available, (2) as an adjunct to the autologous hemodilution management of surgery, or even (3) in radiation therapy or the management of cancer . . . ” (Klein H G (2005) Dev Biol (Basel) 120:45-52).
The above analysis illustrates that the ideal blood substitute was not yet developed. The perfect blood substitute should sustain the patient until hemorrhage will be controlled. Since acellular blood substitute have short circulatory half-lives they should have an ability to stimulate erythropoiesis to compensate the blood loss. To sustain the patient, blood substitute should maximize blood flow and tissue perfusion and therefore, oxygenation. To stimulate erythropoiesis, blood substitute should stabilize HIF-1 alpha under hypoxic and normoxic conditions and by controlling the pro-oxidative and pro-inflammatory reactions should facilitate HIF-1 alpha binding to the EPO-gene. The blood substitute should also not be involved in any pro-apoptotic activity, since a principal function of EPO is to rescue committed erythroid progenitors from apoptosis. These events are necessary to initiate an effective erythropoiesis that in turn will momentarily compensate lost blood with endogenous red blood cells.
A proper delivery of oxygen to ischemic organs is the principal requirement in the regulatory approval of hemoglobin solutions as blood substitute. Therefore, blood substitute that induce vasoconstriction and possess strong pro-oxidative, pro-inflammatory and pro-apoptotic potential could be harmful to the patient and are clinically unacceptable.
The products presently under clinical trial represent the first generation of blood substitute, with effort now directed towards a “new generation” of blood substitute which addresses all of the hemoglobin intrinsic toxicity problems. It is believed that the second-generation products could be used for all clinical indication, including treatment of acute blood loss anemia and trauma.
Since the currently tested blood substitute lack erythropoietic activity, there still exists a need for an improved oxygen carrying solution, which will have the ability to maximize tissue perfusion and oxygenation and stimulate erythropoiesis, thus, replace lost blood with endogenous red blood cells. The stimulation of erythropoiesis by such blood substitute should occur in hypoxic and normoxic conditions. In the case of life-threatening anemia, such blood substitute should serve as initial therapy to maintain tissue oxygenation and secondary therapy to normalize the hematocrit through stimulation of patients' erythropoietic responses through stabilization of HIF-1 alpha and EPO production. This therapy should eliminate the need for an expensive recombinant EPO medication.
In the patent literature there are some indications that stabilization of HIF-1 alpha could provide therapeutic benefits in the treatment of hypoxia related tissue injury. U.S. Pat. No. 6,562,799 to Semenza provides a method for treating a hypoxia- or ischemia-related tissue damage by administering to the subject a therapeutically effective amount of a stable HIF-1 alpha protein. U.S. Pat. No. 6,432,927 to Gregory, et al. provides a method for reducing ischemic tissue damage with the DNA binding domain of a hypoxia inducible factor protein capable of transcriptional activation. Kaelin et al. in U.S. Pat. No. 6,849,718 provides pharmaceutical compositions containing HIF-1 alpha muteins and method of using those compositions to treat hypoxia and ischemic related tissue damage. U.S. Pat. No. 6,838,430 to Arbeit provides the use of stable HIF-1 alpha variants to accelerate wound healing.
These patented methods, however, do not concern the use of hemoglobin based blood substitute in promoting HIF-1 alpha dependent EPO induction, therefore erythropoiesis.
Optimizing oxygen delivery to ischemic tissue and organs and effective stimulation of erythropoietic responses are the most important factors in the regulatory approval of these agents as blood substitute. The blood substitutes of the present invention address the problems discussed above.
2.2. Acute Blood Loss Therapies
There is currently no regulatory approved blood substitute in the United States. As such, blood transfusion is the only reliable means of rapidly restoring blood volume in a subject with acute blood loss. However, there are a number of risks associated with blood transfusion. First, donor blood needs to be tested to determine its suitability for transfusion and compatibility to the recipient. Compatibility testing usually involves (1) ABO typing of donor and recipient blood to prevent transfusion of incompatible red blood cells (RBCs); and (2) Rh typing to determine whether the Rh factor Rh0(D) is present (Rh-positive) or absent (Rh-negative) on the RBCs. The donated blood also needs to be screened to identify unexpected anti-RBC antibodies that can cause hemolytic disease or serious transfusion reaction using for example, direct antiglobulin testing (the direct Coombs' test) and indirect antiglobulin testing (the direct Coombs' test).
Assuming the donor blood is a match for the recipient, many complications can still result due to blood transfusion. For example, hemolysis of donor or recipient RBCs (usually the former) during or after transfusion can result from ABO/Rh incompatibility, incompatible plasma, hemolyzed or fragile RBCs (e.g., by overwarming stored blood or contact with inappropriate IV solutions), or injections of nonisotonic solutions. The reaction is most severe when incompatible donor RBCs are hemolyzed by antibody in the recipient's plasma and can cause breathing difficulty, fever and chills, facial flushing, severe pain (especially in the lumber area), as well as shock that lead to a drop in blood pressure, nausea and vomiting. Allergic reactions to an unknown component in donor blood are also common, usually due to allergens in donor plasma or, less often, to antibodies from an allergic donor. These reactions are usually mild, with urticaria, edema, occasional dizziness, and headache during or immediately after the transfusion, although anaphylaxis may occur in some rare instances. Another complication, though less frequent, is transfusion-related acute lung injury that is caused by anti-white blood cell (WBCs) antibodies in donor plasma that agglutinate and degranulate recipient WBCs within the lungs. Transfusion of large amounts of air into a vein can also cause foaming of blood in the heart with consequent inefficient pumping, leading to heart failure. Graft-vs.-host disease, which can be caused by even small numbers of viable lymphocytes in transfused blood or blood components, can also result from a blood transfusion. There is also the concern of bacterial contamination which may occur due to inadequate aseptic technique during collection or by transient asymptomatic donor. Finally, and most importantly, recipients of blood transfusion will always have the risk of viral disease transmission, including, but not limited to, hepatitis, HIV, cytomegalovirus (CMV), and human T-cell lymphotropic virus type I (HTLV-I) infection.